Abstract: Although [Na.sup.+]-[H.sup.+] exchange (NHE) inhibitors such as methyl-N-isobutyl amiloride (MIA) are known to depress the cardiac function, the mechanisms of their negative inotropic effect are not completely understood. In this study, isolated rat hearts were perfused with MIA to study its action on cardiac performance, whereas isolated subcellular organelles such as sarcolemma, myofibrils, sarcoplasmic reticulum, and mitochondria were treated with MIA to determine its effect on their function. The effect of MIA on intracellular [Ca.sup.2+] mobilization was examined in fura-2-AM-loaded cardiomyocytes. MIA was observed to depress cardiac function in a concentration-dependent manner in HC[O.sub.3.sup.-]-free buffer. On the other hand, MIA had an initial positive inotropic effect followed by a negative inotropic effect in HC[O.sub.3.sup.-]-containing buffer. MIA increased the basal concentration of intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) and augmented the KCl-mediated increase in [[[Ca.sup.2+]].sub.i]. MIA did not show any direct effect on myofibrils, sarcolemma, and sarcoplasmic reticulum ATPase activities; however, this agent was found to decrease the intracellular pH, which reduced the myofibrils [Ca.sup.2+]-stimulated ATPase activity. MIA also increased [Ca.sup.2+] uptake by mitochondria without having any direct effect on sarcoplasmic reticulum [Ca.sup.2+] uptake. In addition, MIA did not protect the hearts subjected to mild [Ca.sup.2+] paradox as well as ischemia-reperfusion-mediated injury. These results suggest that the increase in [[[Ca.sup.2+]].sub.i] in cardiomyocytes may be responsible for the initial positive inotropic effect of MIA, but its negative inotropic action may be due to mitochondrial [Ca.sup.2+] overloading as well as indirect depression of myofibrillar [Ca.sup.2+] ATPase activity. Thus the accumulation of [[[H.sup.+]].sub.i] as well as occurrence of intracellular and mitochondrial [Ca.sup.2+] overload may explain the lack of beneficial effects of MIA in preventing the ischemia-reperfusion-induced myocardial injury.
Key words: sodium-hydrogen exchanger, calcium handling proteins, calcium paradox, ischemia-reperfusion.
Resume : Les inhibiteurs de l'echangeur [Na.sup.+]-[H.sup.+] (NHE) comme le methyl-N-isobutyl amiloride (MIA) sont connus pour diminuer la fonction cardiaque, mais les mecanismes de leur effet inotrope negatif ne sont pas totalement compris. Dans la presente etude, on a perfuse des coeurs isoles de rats avec du MIA pour examiner son action sur la performance cardiaque, et on a traite des organites subcellulaires isoles, sarcolemme, myofibrilles, reticulum sarcoplasmique et mitochondries, avec cet agent pour determiner son effet sur leur fonction. On a examine l'effet du MIA sur la mobilisation du [Ca.sup.2+] intracellulaire dans des cardiomyocytes charges en fura-2-AM. Le MIA a diminue la fonction cardiaque en proportion des concentrations utilisees dans le tampon sans HC[O.sub.3.sup.-]. Par contre, le MIA a eu un effet inotrope positif initial suivi d'un effet inotrope negatif dans le tampon contenant du HC[O.sub.3.sup.-]. Le MIA a augmente la concentration de [Ca.sup.2+] intracellulaire ([[[Ca.sup.2+]].sub.i]) basale et amplifie l'augmentation de la [[[Ca.sup.2+]].sub.i] induite par le KCl. Le MIA n'a pas montre d'effet direct sur les activites ATPases des MF, du SL et du RS; toutefois, cet agent a diminue le pH intracellulaire, ce qui a reduit l'activite ATPase stimulee par le [Ca.sup.2+] dans les MF. Le MIA a aussi augmente la capture de [Ca.sup.2+] par les mitochondries sans avoir d'effet direct sur la capture de [Ca.sup.2+] par le RS. De plus, le MIA n'a pas protege les coeurs soumis a un leger paradoxe calcique et a une lesion d'ischemie reperfusion (I/R). Ces resultats donnent a penser que l'augmentation de la [[[Ca.sup.2+]].sub.i] dans les cardiomyocytes pourrait etre a l'origine de l'effet inotrope positif initial du MIA, mais que son action inotrope negative pourrait etre due a une surcharge de [Ca.sup.2+] mitochondriale et a une diminution indirecte de l'activite [Ca.sup.2+]-ATPase myofibrillaire. Ainsi, l'accumulation de [[[H.sup.+]].sub.I] et la presence d'une surcharge intracellulaire et mitochondriale de [Ca.sup.2+] pourrait expliquer l'absence d'effets benefiques du MIA pour la prevention de la lesion d'I/R myocardique.
Mots-cles: echangeur de sodium-hydrogene, proteines du metabolisme du calcium, paradoxe calcique, ischemie-reperfusion.
[Traduit par la Redaction]
Introduction
The cardiac contractile function is determined mainly by the interaction of [Ca.sup.2+] with myofilament proteins as well as the coordinated activities of sarcolemmal (SL) and sarcoplasmic reticulum (SR) [Ca.sup.2+] cycling proteins (Dhalla et al. 1982; Bers 2002). Various investigators have demonstrated that impairment in energy production at the level of mitochondria and energy utilization mechanisms at the level of myofibrils (MF), SL, and SR are the main factors responsible for the ischemia-reperfusion (I/R)-induced contractile dysfunction (Carrozza et al. 1992; Kusuoka and Marban 1992). The cation transport studies have revealed that the accumulation of intracellular [H.sup.+] by anaerobic metabolism during ischemia activates [Na.sup.+]-[H.sup.+] exchange (NHE), which causes an increase in intracellular [Na.sup.+] with a parallel increase in intracellular [Ca.sup.2+] through the [Na.sup.+]-[Ca.sup.2+] exchanger. These events result in the development of intracellular [Ca.sup.2+] overload and depression in cardiac function (Fig. 1) (Karmazyn et al. 1999). Although a wide variety of experimental studies using various amiloride derivatives as NHE inhibitors have demonstrated the protective effects of these agents in terms of improving I/R-mediated cardiac contractile dysfunction (Meng et al. 1993; Karmazyn 1999), the cardioprotective effects of these agents seem to depend upon the animal models, the time of starting the therapy, dose of the agent, as well as the end point determined (Hurtado and Pierce 2001). In fact, amiloride derivatives have been shown to depress the cardiac function under basal conditions (Pierce et al. 1993). Neither the mechanisms of the cardiodepressant effects under basal conditions nor the exact reasons for the lack of beneficial effects under certain pathophysiological situations are completely understood. The present study was designed to test the hypothesis that inhibition of NHE causes direct or indirect alterations in mechanisms for energy production and energy utilization leading to contractile dysfunction. To study the effect of NHE inhibition on cardiac function, isolated hearts were perfused with a known NHE inhibitor, 5-(N-methyl-N-isobutyl) amiloride (MIA) (Moffat and Karmazyn 1993). In view of the fact that HC[O.sub.3.sup.-] interferes with the action of NHE (Shimada et al. 1996), some experiments were carried out to determine the effect of MIA on cardiac function in HC[O.sub.3.sup.-]-free buffer. The effects of MIA on the activities of various subcellular organelles such as mitochondria, MF, SL, and SR were determined to examine changes in energy production and utilization. To understand the effect of MIA on intracellular [Ca.sup.2+] mobilization, the concentration of intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) was measured in quiescent and KCl-depolarized cardiomyocytes in the absence or presence of this agent. In addition, the hearts were treated with MIA before subjecting to mild [Ca.sup.2+] paradox or I/R injury to investigate the effect of NHE inhibition on cardiac function. Because NHE is known to promote the efflux of [H.sup.+] generated by myocardial metabolism (Putney et al. 2002), pH of isolated cardiomyocytes was measured in the absence and presence of MIA.
[FIGURE 1 OMITTED]
Methods
All experimental protocols were approved by the University of Manitoba Animal Care Committee according to the guidelines of the Canadian Council on Animal Care.
Perfusion of isolated rat heart and experimental protocol
Male Sprague-Dawley rats (250-300 g) were anaesthetized with a mixture of ketamine (90 mg/kg body mass) and xylazine (9 mg/kg). The hearts were quickly excised, mounted on Langendorff apparatus and perfused with Krebs-Henseleit (K-H) buffer gassed with 95% [O.sub.2] : 5% C[O.sub.2], 37[degrees]C, pH 7.4 at a constant flow of 10 mL/min (Saini et al. 2005). The composition of K-H solution was (in mmol/L): 120 NaCl, 4.7 KCl, 1.2 K[H.sub.2]P[O.sub.4], 1.2 MgS[O.sub.4], 25 NaHC[O.sub.3], 1.25 Ca[Cl.sub.2], and 11 glucose. The hearts were electrically stimulated at 300 beats/min via a square wave current of 1.5 ms by using Phipps and Bird stimulator (Richmond, Vir.). The left ventricular systolic pressure (LVSP), the rate of change of pressure development (+dP/dt) and rate of change of pressure decay (-dP/dt) were measured via a transducer (Model 1050 BP-Biopac System Inc., Goleta, Calif.), which was connected with a water-filled latex balloon inserted into the left ventricle (LV). At beginning of the experiment, left ventricular end diastolic pressure (LVEDP) was adjusted to approximately 10 mmHg by …
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